The three-dimensional structure of the wild type and mutants of homeodomains in the NK-2 class bound to an uncommon 18 base-pairDNA consensus segment have been studied. Corresponding transgenic experiments on these single site mutations have been or are being carried out. The interactions responsible for the nucleotide sequence- specific binding have been identified. Mutation analysis was carried out on for positions (7, 35, 41,52, 54, and 56) in the homeodomain. Mutation of position 54 from tyrosine to methionine results in a decrease of one order of magnitude in the binding affinity of the homeodomain for the DNA consensus sequence. Surprisingly, transgenic Drosophila lines with this mutation in position 54 and with the endogenous NK-2genes eliminated are not lethal. Mutation of position 35 from alanine to threonine results in a homeodomain that is unable to adopt a folded conformation free in solution at temperatures down to -5 degrees Celsius. This mutation does result in embryonic lethality. The alanine to threonine mutant homeodomain specifically binds to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower that that of the wild-type homeodomain. Although the three- dimensional structure of the mutant in the DNA bound state has three helices and shows the characteristic helix-turn-helix DNA binding motif similar to that of the wild-type homeodomain, a notable structural distortion in the mutant analog is observed. The wild-type homeodomain forms an unusual i, i-5 hydrogen bond with the backbone amide oxygen of residue 35. In the mutant this amide proton hydrogen bond is altered and the structure of the protein in the region of helix-II is distorted relative to that of the wild-type analog. The tentative conclusion is that mutations that globally lower DNA binding affinity lead to embryonic lethality, whereas mutations that select for a different set of promotor sites likely result in altered phenotypes but are not necessarily lethal. - NMR vnd/NK-2 homeodomain Drosophila three- dimensional structure site mutation